title: Isothermal detection of multiple point mutations by a surface plasmon resonance biosensor with Au nanoparticles enhanced surface-anchored rolling circle amplification.
abstract: In this study, we developed a surface plasmon resonance (SPR) DNA biosensor method using surface-anchored rolling circle amplification (RCA) and Au nanoparticles modified probes (AuNPs) to isothermally detect multiple point mutations associated with drug-resistance in multidrug-resistant Mycobacterium Tuberculosis (MDRTB). A set of probes contains an allele-specific padlock probe (PLP), a capture probe and an AuNPs. The linear PLPs, circularized by ligation upon the recognition of the point mutation on DNA targets, hybridize to the capture probes via the specific tag/anti-tag recognition. Upon recognition each point mutation is identified by locating into the corresponding channel on the chip. Then the immobilized primer (capture probe)-template (circular PLP) complex are amplified isothermally as RCA and further amplified by AuNPs. The RCA products immobilized on the chip surface cause great SPR angle changes consequently. The 5 pM synthetic oligonucleotides and 8.2 pg uL(-1) of genomic DNA from clinical samples can be detected by the method. The positive mutation detection is achieved with a wild-type to mutant ratio of 5000:1. The method was demonstrated by targeting five clinically meaningful mutations in MDRTB. Thirty clinical samples were identified and they were in good agreement with the results from sequencing.
authors.affiliation: Laboratory of Clinical Experimental Base of Biosensor and Microarray, and Center of Molecule and Gene Diagnosis, Southwest Hospital, Third Military Medical University, Chongqing 400038, PR China. xyang_@hotmail.com
authors.forename: Yang
authors.initials: Y
authors.lastname: Xiang
authors.forename: Kun
authors.initials: K
authors.lastname: Deng
authors.forename: Han
authors.initials: H
authors.lastname: Xia
authors.forename: Chunyan
authors.initials: C
authors.lastname: Yao
authors.forename: Qinghai
authors.initials: Q
authors.lastname: Chen
authors.forename: Liqun
authors.initials: L
authors.lastname: Zhang
authors.forename: Zhiyong
authors.initials: Z
authors.lastname: Liu
authors.forename: Weiling
authors.initials: W
authors.lastname: Fu
mesh_terms: D004269:DNA, Bacterial / Q000235:genetics*; D024881:Drug Resistance, Bacterial; D018432:Drug Resistance, Multiple; D006046:Gold / Q000737:chemistry*; D006801:Humans; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D053758:Nanoparticles / Q000737:chemistry*; D009693:Nucleic Acid Hybridization / Q000379:methods; D017354:Point Mutation; D012680:Sensitivity and Specificity; D020349:Surface Plasmon Resonance / Q000379:methods*; D014376:Tuberculosis / Q000382:microbiology
chemical_list: D004269:DNA, Bacterial; D006046:Gold
keywords: Au nanoparticles; Multidrug-resistant Mycobacterium tuberculosis; Multiplex mutation detection; Rolling circle amplification; Surface plasmon resonance